Our current defense against Bacillus anthracis, and other bacterial bioweapons, is based primarily on the use of antibiotic therapies. This plan is flawed considering the possibility that the bacteria used in such weapons may express naturally occurring or engineered resistances to current therapeutic or prophylactic antibiotics. For this reason we are currently developing a novel class of antibacterial agents. Our system is based on the use of phage lysins to provide a rapid and specific killing action against bacterial pathogens of interest, in particular B. anthracis. In addition to offering a previously unavailable method of bacterial killing, phage lysins are primarily attractive in that bacterial resistance to their action cannot be detected, even after extensive attempts. Our laboratory is the first to use these enzymes in their purified form to kill colonizing pathogenic bacteria on mucous membrane surfaces and in blood. The enzymes are specific for the species or strain from which the enzymes were derived, indicating that these enzymes may be used for targeted killing of only the pathogenic bacterium with little to no effect on normal flora bacteria. During these studies we discovered that enzymes with two different specificities for cell wall bonds (i.e., amidase and muramidase) have a synergistic effect in their killing capacity. In our studies with the PlyG phage enzyme from the gamma phage that is specific for B. anthracis, we show that this enzyme is able to kill anthrax bacilli in vitro, reducing 108 bacteria to sterility in two minutes. In vivo, we are able to protect animals from lethal challenge with both a closely related bacillus to B. anthracis as well as B. anthracis. Because of the synergistic effects in these enzymes, this application is designed to identify and develop a combination of enzymes for B. anthracis that attack the four different bonds in the bacillus cell wall. This will ensure a more efficient killing action as well as reduce the possibility of the development of resistance to these enzymes. Phage enzymes will be isolated from phage found in the environment and phage lysogenizing B. anthracis. These enzymes will then be characterized as to their specificity, purified and used in both in vitro and in vivo systems to determine efficacy. Because these enzymes may be an important line of defense against an attack with drug-resistant B. anthracis, having a number of enzymes at our disposal may allow for better decisions as to their use if necessary.